Effects of phase cancellation and receiver aperture size on broadband ultrasonic attenuation for trabecular bone in vitro

Phase cancellation in ultrasound due to large receiver size has been proposed as a contributing factor to the inaccuracy of estimating broadband ultrasound attenuation (BUA), which is used to characterize bone quality. Transducers with aperture size ranging from 2 to 5 mm have been used in previous attempts to study the effect of phase cancellation. However, these receivers themselves are susceptible to phase cancellation because aperture size is close to one center wavelength (about 3 mm at 500 KHz in water). This study uses an ultra small receiver (aperture size: 0.2 mm) in conjunction with a newly developed two-dimensional (2-D) synthetic array system to investigate the effects of phase cancellation and receiver aperture size on BUA estimations of bone tissue. In vitro ultrasound measurements were conducted on 54 trabecular bone samples (harvested from sheep femurs) in a confocal configuration with a focused transmitter and synthesized focused receivers of different aperture sizes. Phase sensitive (PS) and phase insensitive (PI) detections were performed. The results show that phase cancellation does have a significant effect on BUA. The normalized BUA (nBUA) with PS is 8.1% higher than PI nBUA while PI BUA is well correlated with PS BUA. Receiver aperture size also influences the BUA reading for both PI and PS detection and smaller receiver aperture tends to result in higher BUA readings. The results also indicate that the receiver aperture size used in the confocal configuration with PI detection should at least equal the aperture of the transmitter to capture most of the energy redistributed by the interference and diffraction from the trabecular bone.     

Analysis of ultrasound fields in cell culture wells for in vitro ultrasound therapy experiments

Ultrasound is an established therapy method for bone fracture healing, hyperthermia and the ablation of solid tumors. In this new emerging field, ultrasound is further used for microbubble-enhanced drug delivery, gene therapy, sonoporation and thrombolysis. To study selected therapeutic effects in defined experimental conditions, in vitro setups are designed for cell and tissue therapy. However, in vitro studies often lack reproducibility and the successful transfer to other experimental conditions. This is partly because of the uncertainty of the experimental conditions in vitro. In this paper, the ultrasound wave propagation in the most common in vitro ultrasound therapy setups for cell culture wells is analyzed in simulations and verified by hydrophone measurements. The acoustic parameters of the materials used for culture plates and growth media are determined. The appearance and origin of standing waves and ring interference patterns caused by reflections at interfaces is revealed in simulations and measurements. This causes a local maximal pressure amplitude increase by up to the factor of 5. Minor variations of quantities (e.g., growth medium volume variation of 2.56%) increase or decrease the peak rarefaction pressure at a cell layer by the factor of 2. These pressure variations can affect cell therapy results to a large extent. A sealed cell culture well submersed in a water bath provides the best reproducibility and therefore promises transferable therapy results.     

High-resolution ultrasound imaging of human skin in vivo by using three-dimensional ultrasound microscopy

Observing the morphology of human skin is important in the diagnosis of skin cancer and inflammation and in the assessment of skin aging. High-frequency ultrasound imaging provides high spatial resolution of the deep layers of the skin, which cannot be visualized by optical methods. The objectives of the present study were to develop a three-dimensional (3-D) ultrasound microscope and to observe the morphology of normal human skin in vivo. A concave polyvinylidene fluoride transducer with a central frequency of 120 MHz was excited using an electric pulse generated by semiconductor switching. The transducer was scanned two-dimensionally by using two linear motors on the region-of-interest and the ultrasonic reflection was digitized with 2-GHz sampling. Consecutive B-mode images perpendicular to the skin surface were reconstructed to generate multiplanar reconstructed images and 3-D volume-rendering images clearly showing microstructures such as sebaceous glands and hair follicles. The 3-D ultrasound microscope could be used to successfully image the morphology of human skin noninvasively and may provide important information on skin structure.     

Features of in vitro ultrasound biomicroscopic imaging and colonoscopy for detection of colon tumor in mice

The present work tested the capability of ultrasound biomicroscopy (UBM), at 45 MHz, to provide cross-sectional images with appropriate resolution and contrast to detect tumors and determine their penetration depths on the colon of mice, Mus musculus (Linnaeus 1758), treated with carcinogen for colon tumor induction. B-mode images were obtained, in vitro, from each animal (13 treated and 4 untreated) colon opened longitudinally and immersed in saline solution at room temperature. Prior to UBM inspection, all animals were also examined by colonoscopy. The layers of normal colon identified by UBM are: mucosa (hyperechoic), muscularis mucosae (hypoechoic), submucosa (hyperechoic) and muscularis externa (hypoechoic). UBM images of colon lesions presented structures corresponding to tumors (hyperechoic), lymphoid hyperplasia (hypoechoic) and polypoid tumors (hyperechoic). Additionally, tumoral lesion invasion through the colon was also identified. When compared with histopathologic analysis, all colon lesions detected by UBM were confirmed, while colonoscopic findings had two false negatives.     

High-frequency ultrasound for in vivo measurement of colon wall thickness in mice

Mouse models are becoming increasingly important in the study of molecular mechanisms of colorectal disease and in the development of novel therapeutics. To enhance this phase of preclinical research, cost-effective, easy to use noninvasive imaging is required to detect and monitor changes in the colon wall associated with disease pathology. This study investigated the feasibility of using 40-MHz (high frequency) B-mode ultrasound (HF-US) to image the normal mouse colon and measure its thickness in vivo by establishing a robust imaging protocol and conducting a blinded comparison of colon wall thickness (CWT) measurement between and within operators. The in vivo and ex vivo appearance of mouse colon under HF-US revealed distinct patterns. Colon wall thickness was reproducibly and accurately measured using HF-US compared with histology measurement. The technique was more sensitive in detecting changes in CWT in distal than proximal colon as it showed the highest level of inter- and intraoperator reproducibility. Using the protocol described, it is possible to detect changes in thickness of 0.09 mm and 0.25 mm in distal and proximal colon, respectively. In conclusion, HF-US provides an easy to use and noninvasive method to perform anatomical investigations of mouse colon and to monitor changes in CWT.     

Reproducibility of volumetric intravascular ultrasound radiofrequency-based analysis of coronary plaque composition in vivo

Intravascular ultrasound radiofrequency (RF-IVUS) data permit the analysis of coronary plaque composition in vivo and is used as an endpoint of ongoing pharmacological intervention trials. We assessed the reproducibility of volumetric RF-IVUS analyses in mild-to-moderately diseased atherosclerotic human coronary arteries in vivo. A total of 9,212 IVUS analyses on cross-sectional IVUS frames was performed to evaluate the reproducibility of volumetric RF-IVUS measurements in 33 coronary segments with a length of 27 ± 7 mm. For vessel, lumen, and plaque + media volume the relative measurement differences (P = NS for all) were (A = intraobserver comparison, same pullback) −0.40 ± 1.0%; −0.48 ± 1.4%; −0.35 ± 1.6%, (B = intraobserver comparison, repeated pullback) −0.42 ± 1.2%; −0.52 ± 1.8%; −0.43 ± 4.5% (C = interobserver comparison, same pullback) 0.71 ± 1.8%; 0.71 ± 2.2%, and 0.89 ± 5.0%, respectively. For fibrous, fibro-lipidic, calcium, and necrotic-core volumes the relative measurement differences (P = NS for all) were (A) 0.45 ± 2.1%; −1.12 ± 4.9%; −0.84 ± 2.1%; −0.22 ± 1.8%, (B) 1.40 ± 4.1%; 1.26 ± 6.7%; 2.66 ± 7.4%; 0.85 ± 4.4%, and (C) −1.60 ± 4.9%; 3.85 ± 8.2%; 1.66 ± 7.5%, and −1.58 ± 4.7%, respectively. Of note, necrotic-core volume showed on average the lowest measurement variability. Thus, in mild-to-moderate atherosclerotic coronary artery disease the reproducibility of volumetric compositional RF-IVUS measurements from the same pullback is relatively high, but lower than the reproducibility of geometrical IVUS measurements. Measurements from repeated pullbacks and by different observers show acceptable reproducibilities; the volumetric measurement of the necrotic-core shows on average the highest reproducibility of the compositional RF-IVUS measurements     

Effects of transforming growth factor-β subtypes on in vitro cartilage production and mineralization of human bone marrow stromal-derived mesenchymal stem cells

Human bone marrow stromal-derived mesenchymal stem cells (hBMSCs) will differentiate into chondrocytes in response to defined chondrogenic medium containing transforming growth factor-β (TGFβ). Results in the literature suggest that the three mammalian subtypes of TGFβ (TGFβ1, TGFβ2 and TGFβ3) provoke certain subtype-specific activities. Therefore, the aim of our study was to investigate whether the TGFβ subtypes affect chondrogenic differentiation of in vitro cultured hBMSCs differently. HBMSC pellets were cultured for 5 weeks in chondrogenic media containing either 2.5, 10 or 25 ng/ml of TGFβ1, TGFβ2 or TGFβ3. All TGFβ subtypes showed a comparable dose-response curve, with significantly less cartilage when 2.5 ng/ml was used and no differences between 10 and 25 ng/ml. Four donors with variable chondrogenic capacity were used to evaluate the effect of 10 ng/ml of either TGFβ subtype on cartilage formation. No significant TGFβ subtype-dependent differences were observed in the total amount of collagen or glycosaminoglycans. Cells from a donor with low chondrogenic capacity performed equally badly with all TGFβ subtypes, while a good donor overall performed well. After addition of β-glycerophosphate during the last 2 weeks of culture, the expression of hypertrophy markers was analysed and mineralization was demonstrated by alkaline phosphatase activity and alizarin red staining. No significant TGFβ subtype-dependent differences were observed in expression collagen type X or VEGF secretion. Nevertheless, pellets cultured with TGFβ1 had significantly less mineralization than pellets cultured with TGFβ3. In conclusion, this study suggests that TGFβ subtypes do affect terminal differentiation of in vitro cultured hBMSCs differently.     

New method for quantifying and correcting underestimated cardiac Doppler blood flow velocities: in vitro and in vivo studies

This study was aimed to quantify the underestimation of cardiac Doppler measurements and to explore a method for correction. A dual pulse wave (PW)/Doppler tissue imaging (DTI) mode echocardiographic technique was used in the in vitro and in vivo studies. In the in vitro experiment, we have demonstrated how cardiac valvular motion might interfere with blood velocity estimation using conventional Doppler. When examining the participants, we observed that adding valvular annulus velocity to determine the relative velocity between blood and valvular annulus would result in an increment of 9.3 ± 1.3 cm/s and 6.3 ± 0.9 cm/s for aortic and pulmonary blood flow, 12.8 ± 1.9 cm/s and 8.9 ± 1.4 cm/s for mitral E and A wave, 12.9 ± 1.8 cm/s and 10.2 ± 2.4 cm/s for tricuspid E and A wave. The underestimations of the Doppler measurements markedly influence the hemodynamic parameters commonly used in the clinical practices and researches. This study provides a quantitative method for the correction and would make the Doppler measurement accurate.     

Effects of peripherally restricted κ opioid receptor agonists on pain-related stimulation and depression of behavior in rats

κ opioid receptor agonists that do not readily cross the blood-brain barrier are peripherally restricted and distribute poorly to the central nervous system after systemic administration. Peripherally restricted κ agonists have promise as candidate analgesics, because they may produce antinociception mediated by peripheral κ receptors more potently than they produce undesirable sedative and psychotomimetic effects mediated by central κ receptors. The present study used assays of pain-related stimulation and depression of behavior in rats to compare effects of 1) two peripherally restricted κ agonists [the tetrapeptide D-Phe-D-Phe-D-Ile-D-Arg-NH(2) (ffir) and the nonpeptidic compound ((R,S)-N-[2-(N-methyl-3,4-dichlorophenylacetamido)-2-(3-carboxyphenyl)-ethyl]pyrrolidine hydrochloride (ICI204448)], 2) a centrally penetrating κ agonist (salvinorin A), and 3) several reference drugs, including a nonsteroidal anti-inflammatory drug (NSAID; ketoprofen). Intraperitoneal injection of dilute lactic acid served as a noxious stimulus to stimulate a stretching response and depress intracranial self-stimulation (ICSS) maintained by the delivery of electrical brain stimulation to the medial forebrain bundle. Acid-stimulated stretching was blocked by ketoprofen, the peripherally restricted κ agonists, and salvinorin A. However, acid-induced depression of ICSS was blocked only by ketoprofen. The peripherally restricted κ agonists had little effect, and salvinorin A exacerbated acid-induced depression of ICSS. These results suggest that peripherally restricted κ agonists may be safer than centrally penetrating κ agonists but less efficacious than NSAIDS or μ opioid receptor agonists to block pain-related depression of behavior; however, the peripheral selectivity of ffir and ICI204448 is limited, and future studies with κ agonists capable of greater peripheral selectivity are warranted.     

Regulation of κ-opioid receptor signaling in peripheral sensory neurons in vitro and in vivo

There is considerable interest in understanding the regulation of peripheral opioid receptors to avoid central nervous system side effects associated with systemically administered opioid analgesics. Here, we investigated the regulation of the κ-opioid receptor (KOR) on rat primary sensory neurons in vitro and in a rat model of thermal allodynia. Under basal conditions, application of the KOR agonist trans-(1S,2S)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide hydrochloride hydrate (U50488) did not inhibit adenylyl cyclase (AC) activity nor release of calcitonin gene-related peptide (CGRP) in vitro and did not inhibit thermal allodynia in vivo. However, after 15-min pretreatment with bradykinin (BK), U50488 became capable of inhibiting AC activity, CGRP release, and thermal allodynia. Inhibition of AC by 5-hydroxytryptamine 1 or neuropeptide Y(1) receptor agonists and stimulation of extracellular signal-regulated kinase activity by U50488 did not require BK pretreatment. The effect of U50488 in BK-primed tissue was blocked by the KOR antagonist nor-binaltorphimine both in vitro and in vivo. The effect of BK in vitro was blocked by either indomethacin or bisindolylmaleimide, suggesting that an arachidonic acid (AA) metabolite and protein kinase C (PKC) activation mediate BK-induced regulation of the KOR system. Furthermore, the effect of U50488 in BK-treated tissue was blocked by a soluble integrin-blocking peptide (GRGDSP), but not the inactive reverse sequence peptide (GDGRSP), suggesting that, in addition to AA and PKC, RGD-binding integrins participate in the regulation of KOR signaling in response to U50488. Understanding the mechanisms by which peripheral KOR agonist efficacy is regulated may lead to improved pharmacotherapy for the treatment of pain with reduced adverse effects.     

Dosing optimization of meropenem based on a pharmacokinetic analysis in patients receiving hemodiafiltration and an in vitro model

The purpose of this study was to estimate the in vivo pharmacokinetics of meropenem during intermittent-infusion hemodiafiltration (I-HDF) and clarify its optimal dosage and dosing interval in patients receiving I-HDF. The clearance of meropenem by online hemodiafiltration (OL-HDF) and I-HDF was predicted using an in vitro system and assessed to establish whether the results obtained are applicable to clinical cases. In the in vivo study, the mean volume of distribution (Vd), non-I-HDF clearance (CL_non-I-HDF), and I-HDF clearance (CL_I-HDF) were 15.80 ± 3.59 l, 1.05 ± 0.27 l/h, and 5.78 ± 1.03 l/h. Dosing regimens of 0.25 g once daily for a MIC of 8 μg/ml and of 0.5 g once daily for a MIC of 16 μg/ml achieved 40% T > MIC. In the in vitro and in vivo studies, observed CL_HDF was similar to predictive CL_HDF (= C_f/C_p × (Q_D + Q_SUB)). In conclusion, adjustments to the dose and interval of meropenem were developed based on the presumed susceptibility of pathogens to meropenem in patients receiving I-HDF. We suggest 0.5 g once daily as an appropriate regimen for empirical treatment.     

Study on roles of gap junctional intercellular communication in low dose parathyroid hormones-induced bone anabolism in vitro

AIM:To investigate mediating and reguatory effects of osteoblastic gap junctional interecllular communication(GJIC)on low-dose parathyroid hor-mones(PTH)-stimulated bone formation activties in vitro.METHODS:Rat calvarial osteoblasts(ROBs) in cultures wer divided into three groups ac-cording to the different mode of exposure.Group A:vehicle (sodium acetate,SA)-treated group;GroupB:1&;#215;10^-8mol/L hPTH(1-34)intermittent exposure group;GroupC:1&;#215;10^-8mol/LhPTH(1-34)+1&;#215;10^-7mol/L TPA exposure group.48h incubation cycles in three groups were repeated for eight times.GJIC and mineralized bone nodules formation in three groups were detected using Lucifer Yellow(LY) scrape loading dye transfer(SLDT) and mineralized nodule staining together with nodule index,respectively.RESULTS:At various measuring time points of SA&;#215;6h+TPA&;#215;1h in group C,LY(+) cell numbers were 6.8&;#177;2.5,19.5&;#177;6.5,14.0&;#177;3.6 and 5.7&;#177;2.4,respectively.Diffusion and transfer of LY fluorescent probe was much more noticeably discerned in group B than in group A and C(P&;lt;0.01).Mineralized bone nodule indices were 45.2&;#177;12.5,88.0&;#177;15.3and 38.5&;#177;17.9 in group A,B and C respectively.Bone formation activity was much better revealed in group B than in group A and C(P&;lt;0.01）,whereas no statistically significant difference of bone formation activities were found in group A compared with group C(P=0.465).CONCLUSION:Mediations and regulations of the coordinating signals in osteoblastic network via GJIC essentially contribute to PTH-stimulated bone anabolism.However,disruption of GJIC not only hinders osteoblastic intercellular coordination but also frustrated PTH-induced bone formation activities in vitro.Therefor,GJIC may evidently play imprtant roles in regulations on low-dose PTH-induced bone formation.     

Effects of surface electrical stimulation on the muscle–tendon junction of spastic gastrocnemius in stroke patients

Purpose:?The purpose of this study was to explore the effects of spasticity suppression by surface electrical stimulation (ES) on the muscle–tendon junction of spastic gastrocnemius muscles in stroke.

Methods:?Twenty-four neurologically stable stroke patients (aged 41?–?69 years, 12?–?35 months post-stroke), with spasticity graded 2 or 3 on the modified Ashworth scale, were recruited and divided into two groups. In the ES group, each patient received 20?min of surface ES once daily, 6 days per week for 1 month. In the control group, ES was used with stimulation intensity kept at zero. To evaluate the therapeutic effect, the modified Ashworth scale, Fmax/Mmax ratio, H-reflex latency, H-reflex recovery curve, and the 10-m walking time were tested before and after the 1-month treatment.

Results:?In the ES group, the modified Ashworth Scale showed a trend toward reduced spasticity after 1 month of treatment. The Fmax/Mmax ratio decreased from 8.10%?±?4.84% to 4.00%?±?1.36%; the H-reflex latency increased from 28.87?±?2.45?ms to 29.40?±?2.57?ms; the H-reflex recovery curves indicated a downward shift; and the 10-metre walking time significantly decreased after ES. In the control group, none of the measures showed a statistically significant change.

Conclusions:?In the study, we demonstrated a way to suppress spasticity at a metameric site and to increase walking speed effectively by applying surface ES on the muscle–tendon junction of spastic gastrocnemius muscles.     

Determination of intracellular and extracellular β-lactamase activities of Pseudomonas aeruginosa after exposure to β-lactams in vitro and in vivo

β-Lactamase production in Pseudomonas aeruginosa was determined in in-vitro models and in rat pouch infection models after exposure to ceftazidime, imipenem, and piperacillin. Exposure of 28 P. aeruginosa strains to 1/4 minimum inhibitory concentration (MIC) of ceftazidime, imipenem, and piperacillin for 24 h enhanced intracellular β-lactamase activities in 14, 22, and 6 strains, respectively, of the 28 clinical strains tested, and enhanced extracellular β-lactamase activities which were not detected without exposure to antibiotics, in 7, 23, and 1 of the 28 strains, respectively. Extracellular β-lactamase activity from P. aeruginosa S-1278, producing an inducible β-lactamase, scarcely increased after exposure to ceftazidime and piperacillin 24 h after incubation, while the activity increased after exposure to imipenem over the range of 1/8 to 8 MIC. In the rat granuloma pouch models infected with P. aeruginosa S-1278, ceftazidime and piperacillin, after single administration (20 mg/kg) and serial administration (20 mg/kg per day × 3 days), did not enhance extracellular β-lactamase activities. However, the activities were enhanced with single and serial administrations of imipenem, and levels over 10 mU/ml were detected until the third day. The β-lactamase activity, similar to the activity found in rat pouches after serial administration of imipenem, inactivated various cephalosporins. In conclusion, extracellular β-lactamase activity was detected both in vitro and in vivo after exposure to a good inducer, and extracellular β-lactamase remained at infection site at levels that could inactivate cephalosporins. Received: May 1, 2000 / Accepted: August 9, 2000